Effect of chronic intermittent hypoxia (CIH) on neuromuscular junctions and mitochondria in slow- and fast-twitch skeletal muscles of mice—the role of iNOS

Abstract

Background Obstructive sleep apnea (OSA) imposes vascular and metabolic risks through chronic intermittent hypoxia (CIH) and impairs skeletal muscle performance. As studies addressing limb muscles are rare, the reasons for the lower exercise capacity are unknown. We hypothesize that CIH-related morphological alterations in neuromuscular junctions (NMJ) and mitochondrial integrity might be the cause of functional disorders in skeletal muscles. Methods Mice were kept under 6 weeks of CIH (alternating 7% and 21% O2 fractions every 30 s, 8 h/day, 5 days/week) compared to normoxia (NOX). Analyses included neuromuscular junctions (NMJ) postsynaptic morphology and integrity, fiber cross-sectional area (CSA) and composition (ATPase), mitochondrial ultrastructure (transmission-electron-microscopy), and relevant transcripts (RT-qPCR). Besides wildtype (WT), we included inducible nitric oxide synthase knockout mice (iNOS−/−) to evaluate whether iNOS is protective or risk-mediating. Results In WT soleus muscle, CIH vs. NOX reduced NMJ size (− 37.0%, p < 0.001) and length (− 25.0%, p < 0.05) together with fiber CSA of type IIa fibers (− 14%, p < 0.05) and increased centronucleated fiber fraction (p < 0.001). Moreover, CIH vs. NOX increased the fraction of damaged mitochondria (1.8-fold, p < 0.001). Compared to WT, iNOS−/− similarly decreased NMJ area and length with NOX (− 55%, p < 0.001 and − 33%, p < 0.05, respectively) or with CIH (− 37%, p < 0.05 and − 29%, p < 0.05), however, prompted no fiber atrophy. Moreover, increased fractions of damaged (2.1-fold, p < 0.001) or swollen (> 6-fold, p < 0.001) mitochondria were observed with iNOS−/− vs. WT under NOX and similarly under CIH. Both, CIH- and iNOS−/− massively upregulated suppressor-of-cytokine-signaling-3 (SOCS3) > 10-fold without changes in IL6 mRNA expression. Furthermore, inflammatory markers like CD68 (macrophages) and IL1β were significantly lower in CIH vs. NOX. None of these morphological alterations with CIH- or iNOS−/− were detected in the gastrocnemius muscle. Notably, iNOS expression was undetectable in WT muscle, unlike the liver, where it was massively decreased with CIH. Conclusion CIH leads to NMJ and mitochondrial damage associated with fiber atrophy/centronucleation selectively in slow-twitch muscle of WT. This effect is largely mimicked by iNOS−/− at NOX (except for atrophy). Both conditions involve massive SOCS3 upregulation likely through denervation without Il6 upregulation but accompanied by a decrease of macrophage density especially next to denervated endplates. In the absence of muscular iNOS expression in WT, this damage may arise from extramuscular, e.g., motoneuronal iNOS deficiency (through CIH or knockout) awaiting functional evaluation.

Effects of Monoamino-Oxidase-A (MAO-A) Inhibition on Skeletal Muscle Inflammation and Wasting through Pancreatic Ductal Adenocarcinoma in Triple Transgenic Mice

“…Using TEM, the ultrastructural integrity of gastrocnemius and soleus muscle mitochondria was studied at 25,000-fold magnification as previously described [ 56 ], in 5–8 mice per WT, Ca, WTHH, or CaHH group and >10 images per animal. Mitochondrial ultrastructure was categorized as ‘damaged’ in the case of >50% loss of the cristae, and/or >50% disruption of the outer membrane, or as ‘normal’ otherwise.…”

Severely Damaged Freeze-Injured Skeletal Muscle Reveals Functional Impairment, Inadequate Repair, and Opportunity for Human Stem Cell Application

“…The morphometric evaluation of muscle tissue changes was performed on digital images captured after scanning H&E and Sirius Red stained slides with an Aperio scanner (version 12.3.3: Leica Biosystems, Buccinasco, Milan, Italy). Ten selected images at 20× of H&E-stained slides were analyzed to measure the crosssectional area of muscle cells (CSA, µm 2 , used to evaluate muscle fiber sizes based on fiber area in cross-section) using computerized imaging software (ImageJ-win32, NIH, Bethesda, MD, USA) in 100 muscle cells transversally sectioned and this was expressed as the mean [27]. The consecutive muscle tissue sections (3 µm) stained with Sirius Red were used for morphometric analysis of red-stained interstitial collagen volume fraction with computerized imaging software (Image J-win32, NIH, Bethesda, MD, USA) and expressed as a percentage [28].…”

Acetylome analyses provide novel insights into the effects of chronic intermittent hypoxia on hippocampus-dependent cognitive impairment

“…Moreover, OSA causes repetitive fluctuations in blood oxygen saturation and leads to chronic intermittent hypoxia (CIH). Over the past two decades, the prevalence of OSA has doubled ( Bannow et al, 2022 ). Cognitive and behavioral scales have indicated OSA-induced abnormalities in learning, memory, and cognition ( Hunter et al, 2016 ; Labarca et al, 2020 ; Osorio et al, 2022 ).…”